Medical and ethical challenges in the case of a prenatally undiagnosed massive congenital brain tumor.

Fetal and neonatal brain tumors are rare. Prenatal ultrasound aids early tumor detection. Nonetheless, we encountered a preterm neonate born at 32 weeks gestation with a massive supratentorial glioma, which was undetected on ultrasound at 19-6/7 weeks gestation. The patient presented at birth with unanticipated massive macrocephaly. Resuscitation and stabilization were difficult, but the medical team felt that futility of care was not established and opted to transfer the baby to an academic center for further imaging and specialist consultations. Diagnosis of an extensive, inoperable tumor was confirmed and support withdrawn. Postmortem histologic examination and immunohistochemical stains identified the majority of tumor cells as glial in origin. This case report illustrates well how a severe and potentially fatal anomaly, which remained undetected prenatally, presented the medical team and family with multiple medical, ethical and emotional challenges at birth; decisions regarding futility of care in the neonatal transport setting are difficult.

Schizophrenia-risk variant rs6994992 in the neuregulin-1 gene on brain developmental trajectories in typically developing children.

The neuregulin-1 (NRG1) gene is one of the best-validated risk genes for schizophrenia, and psychotic and bipolar disorders. The rs6994992 variant in the NRG1 promoter (SNP8NRG243177) is associated with altered frontal and temporal brain macrostructures and/or altered white matter density and integrity in schizophrenic adults, as well as healthy adults and neonates. However, the ages when these changes begin and whether neuroimaging phenotypes are associated with cognitive performance are not fully understood. Therefore, we investigated the association of the rs6994992 variant on developmental trajectories of brain macro- and microstructures, and their relationship with cognitive performance. A total of 972 healthy children aged 3-20 years had the genotype available for the NRG1-rs6994992 variant, and were evaluated with magnetic resonance imaging (MRI) and neuropsychological tests. Age-by-NRG1-rs6994992 interactions and genotype effects were assessed using a general additive model regression methodology, covaried for scanner type, socioeconomic status, sex and genetic ancestry factors. Compared with the C-carriers, children with the TT-risk-alleles had subtle microscopic and macroscopic changes in brain development that emerge or reverse during adolescence, a period when many psychiatric disorders are manifested. TT-children at late adolescence showed a lower age-dependent forniceal volume and lower fractional anisotropy; however, both measures were associated with better episodic memory performance. To our knowledge, we provide the first multimodal imaging evidence that genetic variation in NRG1 is associated with age-related changes on brain development during typical childhood and adolescence, and delineated the altered patterns of development in multiple brain regions in children with the T-risk allele(s).

Common DNA methylation alterations in multiple brain regions in autism.

Autism spectrum disorders (ASD) are increasingly common neurodevelopmental disorders defined clinically by a triad of features including impairment in social interaction, impairment in communication in social situations and restricted and repetitive patterns of behavior and interests, with considerable phenotypic heterogeneity among individuals. Although heritability estimates for ASD are high, conventional genetic-based efforts to identify genes involved in ASD have yielded only few reproducible candidate genes that account for only a small proportion of ASDs. There is mounting evidence to suggest environmental and epigenetic factors play a stronger role in the etiology of ASD than previously thought. To begin to understand the contribution of epigenetics to ASD, we have examined DNA methylation (DNAm) in a pilot study of postmortem brain tissue from 19 autism cases and 21 unrelated controls, among three brain regions including dorsolateral prefrontal cortex, temporal cortex and cerebellum. We measured over 485,000 CpG loci across a diverse set of functionally relevant genomic regions using the Infinium HumanMethylation450 BeadChip and identified four genome-wide significant differentially methylated regions (DMRs) using a bump hunting approach and a permutation-based multiple testing correction method. We replicated 3/4 DMRs identified in our genome-wide screen in a different set of samples and across different brain regions. The DMRs identified in this study represent suggestive evidence for commonly altered methylation sites in ASD and provide several promising new candidate genes.

Genome-wide association study of shared components of reading disability and language impairment.

Written and verbal languages are neurobehavioral traits vital to the development of communication skills. Unfortunately, disorders involving these traits-specifically reading disability (RD) and language impairment (LI)-are common and prevent affected individuals from developing adequate communication skills, leaving them at risk for adverse academic, socioeconomic and psychiatric outcomes. Both RD and LI are complex traits that frequently co-occur, leading us to hypothesize that these disorders share genetic etiologies. To test this, we performed a genome-wide association study on individuals affected with both RD and LI in the Avon Longitudinal Study of Parents and Children. The strongest associations were seen with markers in ZNF385D (OR = 1.81, P = 5.45 × 10(-7) ) and COL4A2 (OR = 1.71, P = 7.59 × 10(-7) ). Markers within NDST4 showed the strongest associations with LI individually (OR = 1.827, P = 1.40 × 10(-7) ). We replicated association of ZNF385D using receptive vocabulary measures in the Pediatric Imaging Neurocognitive Genetics study (P = 0.00245). We then used diffusion tensor imaging fiber tract volume data on 16 fiber tracts to examine the implications of replicated markers. ZNF385D was a predictor of overall fiber tract volumes in both hemispheres, as well as global brain volume. Here, we present evidence for ZNF385D as a candidate gene for RD and LI. The implication of transcription factor ZNF385D in RD and LI underscores the importance of transcriptional regulation in the development of higher order neurocognitive traits. Further study is necessary to discern target genes of ZNF385D and how it functions within neural development of fluent language.

Social impairments in Rett syndrome: characteristics and relationship with clinical severity.

BACKGROUND: While behavioural abnormalities are fundamental features of Rett syndrome (RTT), few studies have examined the RTT behavioural phenotype. Most of these reports have focused on autistic features, linked to the early regressive phase of the disorder, and few studies have applied standardised behavioural measures. We used a battery of standardised measures of behaviour and functioning to test the following hypotheses: (1) autistic behaviour is prominent throughout childhood in RTT; (2) autistic features are more salient in individuals with milder presentation; (3) severity of autistic behaviour is associated with a wider range of behavioural problems; and (4) specific MECP2 mutations are linked to more severe autistic behaviour. METHODS: Eighty MECP2 mutation-positive girls with RTT (aged 1.6-14.9 years) were administered: (1) the Screen for Social Interaction (SSI), a measure of autistic behaviour suited for individuals with severe communication and motor impairment; (2) the Rett Syndrome Behaviour Questionnaire (RSBQ), covering a wide range of abnormal behaviours in RTT; (3) the Vineland Adaptive Behavior Scales (VABS); and (4) a modified version of the Rett Syndrome Severity Scale (RSSS). Regression analyses examined the predictive value of age and RSSS on autistic behaviour and other behavioural abnormalities. T-tests further characterised the behavioural phenotype of individual MECP2 mutations. RESULTS: While age had no significant effect on SSI or RSBQ total scores in RTT, VABS Socialization and Composite scores decreased over time. Clinical severity (i.e. RSSS) also increased with age. Surprisingly, SSI performance was not related to either RSSS or VABS Composite scores. Autistic behaviour was weakly linked with the RSBQ Hand behaviour factor scores, but not with the RSBQ Fear/Anxiety factor. Clinical (neurological) severity did not predict RSBQ scores, as evidenced by the analysis of individual MECP2 mutations (e.g. p.R106W, p.R270X and p.R294X). CONCLUSIONS: Our data suggest that in RTT, autistic behaviour persists after the period of regression. It also demonstrated that neurological and behavioural impairments, including autistic features, are relatively independent of one another. Consistent with previous reports of the RTT phenotype, individual MECP2 mutations demonstrate complex associations with autistic features. Evidence of persistent autistic behaviour throughout childhood, and of a link between hand function and social skills, has important implications not only for research on the RTT behavioural phenotype, but also for the clinical management of the disorder.

Autism spectrum disorder in Down syndrome: cluster analysis of Aberrant Behaviour Checklist data supports diagnosis.

BACKGROUND: The diagnostic validity of autism spectrum disorder (ASD) based on Diagnostic and Statistical Manual of Mental Disorders (DSM) has been challenged in Down syndrome (DS), because of the high prevalence of cognitive impairments in this population. Therefore, we attempted to validate DSM-based diagnoses via an unbiased categorisation of participants with a DSM-independent behavioural instrument. METHODS: Based on scores on the Aberrant Behaviour Checklist - Community, we performed sequential factor (four DS-relevant factors: Autism-Like Behaviour, Disruptive Behaviour, Hyperactivity, Self-Injury) and cluster analyses on a 293-participant paediatric DS clinic cohort. The four resulting clusters were compared with DSM-delineated groups: DS + ASD, DS + None (no DSM diagnosis), DS + DBD (disruptive behaviour disorder) and DS + SMD (stereotypic movement disorder), the latter two as comparison groups. RESULTS: Two clusters were identified with DS + ASD: Cluster 1 (35.1%) with higher disruptive behaviour and Cluster 4 (48.2%) with more severe autistic behaviour and higher percentage of late onset ASD. The majority of participants in DS + None (71.9%) and DS + DBD (87.5%) were classified into Cluster 2 and 3, respectively, while participants in DS + SMD were relatively evenly distributed throughout the four clusters. CONCLUSIONS: Our unbiased, DSM-independent analyses, using a rating scale specifically designed for individuals with severe intellectual disability, demonstrated that DSM-based criteria of ASD are applicable to DS individuals despite their cognitive impairments. Two DS + ASD clusters were identified and supported the existence of at least two subtypes of ASD in DS, which deserve further characterisation. Despite the prominence of stereotypic behaviour in DS, the SMD diagnosis was not identified by cluster analysis, suggesting that high-level stereotypy is distributed throughout DS. Further supporting DSM diagnoses, typically behaving DS participants were easily distinguished as a group from those with maladaptive behaviours.

Investigating genotype-phenotype relationships in Rett syndrome using an international data set.

BACKGROUND: Rett syndrome is an uncommon neurodevelopmental disorder with an incidence of 1:9,000 live female births. The principal genetic cause was first reported in 1999 when the association with mutations in the methyl-CpG-binding protein 2 (or MECP2) gene was identified. This study uses data from a large international database, InterRett, to examine genotype-phenotype relationships and compares these with previous findings in a population-based cohort. METHOD: The data set for these analyses was derived from a subset of InterRett cases with subject information collected from the family, the clinician, or both. Individual phenotypic characteristics and clinical severity using three scales were compared among those with eight known recurrent pathogenic MECP2 mutations as well as those with C-terminal deletions (n = 272). RESULTS: Overall, p.R270X and p.R255X were the most severe and p.R133C and p.R294X were the mildest mutations. Significant differences by mutation were seen for individual phenotypic characteristics such as hand use, ambulation, and language. CONCLUSIONS: This multicenter investigation into the phenotypic correlates of MECP2 mutations in Rett syndrome has provided a greater depth of understanding than hitherto available about the specific phenotypic characteristics associated with commonly occurring mutations. Although the modifying influence of X inactivation on clinical severity could not be included in the analysis, the findings confirm clear genotype-phenotype relationships in Rett syndrome and show the benefits of collaboration crucial to effective research in rare disorders.

Selective cerebral volume reduction in Rett syndrome: a multiple-approach MR imaging study.

BACKGROUND AND PURPOSE: Previous studies have examined volumetric abnormalities in Rett syndrome (RTT), using MR imaging and focusing on selective changes. However, these studies preceded the identification of MECP2 as the gene mutated in most RTT cases. We studied regional brain volume changes as noted by MR imaging in girls with RTT who had mutations in the MECP2 gene and more or less severe clinical outcomes to further characterize the neuroanatomy of RTT and its correlations with clinical severity. MATERIALS AND METHODS: Complementary semiautomated Talairach- and voxel-based approaches were used to study spoiled gradient-recalled acquisition sequence MR imaging scans from 23 girls with MECP2 mutations/RTT, including a pair of discordant monozygotic twins and 25 age-matched control girls. Both absolute and relative volumetric changes were examined to account for the well-documented global reduction in brain volume seen in RTT. RESULTS: Absolute volumetric reductions were observed throughout the brain in RTT. Selective/relative decreases in parietal lobe gray matter, particularly in the dorsal parietal region, and mild, diffuse reductions in cortical white matter were observed in the RTT group compared with control subjects. In girls with RTT and a more severe phenotype, anterior frontal lobe volumes were relatively more reduced. Twin comparisons revealed selective preservation of the occipital cortex. CONCLUSION: Selective reductions of dorsal parietal gray matter and preservation of the occipital cortex seem to be basic neuroanatomic features of RTT, whereas preferential reduction of the anterior frontal lobe appears to be a correlate of clinical severity in this disorder. The most affected brain regions include those that may underlie key functional deficits observed in RTT.

Gross motor profile in rett syndrome as determined by video analysis.

Movement impairment is a fundamental but variable component of the Rett syndrome phenotype. This study used video supplemented by parent report data to describe the gross motor profile in females with Rett syndrome (n=99) and to investigate the impact of age, genotype, scoliosis and hand stereotypies. Factor analysis enabled the calculation of general and complex gross motor skills scores. Most subjects were able to sit, slightly less than half were able to walk and a minority were able to transfer without assistance. General gross motor skills declined with age and were poorer in those who had surgically treated scoliosis but not conservatively managed scoliosis. Complex gross motor skills did not decline with age and were better in those without scoliosis. Those with a p.R133C, p.R294X, or a p.R255X mutation appear to have better motor skills overall than those with a p.R270X or large deletion mutation. Motor scores were not related to the frequency of hand stereotypies. This information is useful for the clinician and family when planning support strategies and interventions.

Development of a video-based evaluation tool in Rett syndrome.

This paper describes the development of a video-based evaluation tool for use in Rett syndrome (RTT). Components include a parent-report checklist, and video filming and coding protocols that contain items on eating, drinking, communication, hand function and movements, personal care and mobility. Ninety-seven of the 169 families who initially agreed to participate returned a videotape within 8 months of the first request. Subjects whose videos were returned had a similar age profile to those who did not provide a video but were more likely to have classical than atypical RTT. Evidence of the content and social validity and inter-rater reliability on 11 videos is provided. Video may provide detailed, objective assessment of function and behaviour in RTT.

Early progressive encephalopathy in boys and MECP2 mutations.

MECP2 mutations mainly occur in females with Rett syndrome. Mutations have been described in 11 boys with progressive encephalopathy: seven of nine with affected sisters and two de novo. The authors report four de novo occurrences: three pathogenic and one potentially pathogenic. Common features include failure to thrive, respiratory insufficiency, microcephaly, and abnormal motor control. MECP2 mutations should be assessed in boys with progressive encephalopathy and one or more of respiratory insufficiency, abnormal movements or tone, and intractable seizures.

Cycloxygenase-2 activity promotes cognitive deficits but not increased amyloid burden in a model of Alzheimer's disease in a sex-dimorphic pattern.

Administration of non-steroidal anti-inflammatory agents reduces the risk of developing Alzheimer's disease in normal aging populations, an effect that may occur from inhibition of the cyclooxygenases, the rate-limiting enzymes in the formation of prostaglandins. In this study, we investigated whether increased activity of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, potentiates disease progression in a transgenic mouse model of Alzheimer's disease. To study the functional effects of COX-2 activity, male and female bigenic mice (amyloid precursor protein with Swedish mutation [APPswe]-presenilin-1 protein with deletion of exon 9 [PS1dE9] and trigenic COX-2/APPswe-PS1dE9) were behaviorally tested +/-administration of the selective COX-2 inhibitor celecoxib. Behavioral testing included a three-trial Y maze that measures spatial working and recognition memories and an open field task that tested levels of hyperactivity. Overexpression of COX-2 in APPswe-PS1dE9 mice resulted in specific deficits in spatial working memory in female but not male mice. These sex-specific deficits were abolished by pharmacological inhibition of COX-2 activity. Importantly, COX-2-associated deficits were dependent on co-expression of all three transgenes since COX-2 single transgenic and APPswe-PS1dE9 bigenic mice showed normal memory. Quantification of amyloid plaque load and total Abeta 40 and 42 peptides did not reveal significant differences in trigenic versus bigenic mice treated with either vehicle or celecoxib. Taken together, these data indicate an interaction between the effects of COX-2 and Abeta peptides on cognition that occurs in a sex-specific manner in the absence of significant changes in amyloid burden. These findings suggest that pathological activation of COX-2 may potentiate the toxicity of Abeta peptides, particularly in females, without significantly affecting Abeta accumulation.

Methyl-CpG-binding protein 2 is localized in the postsynaptic compartment: an immunochemical study of subcellular fractions.

Methyl-CpG-binding protein 2 is a characteristic member of the methyl-CpG-binding protein family of transcription regulators. In conjunction with Sin3, MeCP2 recruits class I histone deacetylases to methyl-CpG regions to suppress transcription. Rett syndrome, a disorder characterized by mental retardation and autistic features, is associated in a majority of cases with mutations within the coding region of the MeCP2 gene. Considering that defective MeCP2 has mainly been related to Rett syndrome and other neurologic manifestations, we examined methyl-CpG-binding protein 2 cellular and subcellular compartmentalization in normal brain by immunochemical methods. Methyl-CpG-binding protein 2 immunoreactivity is present mainly in neurons; while the few immunostained glia show label confined to nuclei, many neurons also show slight perikaryal staining. Using well-characterized tissue fractions, we found that methyl-CpG-binding protein 2 but not Sin3 is found in both nuclear and postsynaptic compartments. This novel extranuclear localization is not unique to methyl-CpG-binding protein 2, since it has been previously reported for other transcription regulators such as c-Fos. These findings support the concept that methyl-CpG-binding protein 2 may link synaptic activity and transcriptional regulation in neurons.

Megalencephaly in NF1: predominantly white matter contribution and mitigation by ADHD.

BACKGROUND: Megalencephaly is a frequent CNS manifestation in neurofibromatosis type 1 (NF1); however, its tissue composition, modification by attention deficit hyperactivity disorder (ADHD), and relationship with unidentified bright objects (UBO) remain controversial. METHODS: Eighteen male patients with NF1, seven of whom had ADHD (NF1+ADHD), were compared with 18 age- and sex-matched controls in terms of MRI-, Talairach-based brain, cerebral, lobar, and sublobar gray and white matter volumes. Twelve subjects with NF1 had UBO in the centrencephalic region, whereas six had no UBO or exclusively infratentorial lesions. RESULTS: Patients with NF1 without ADHD (NF1-pure) had the largest total cerebral, gray, and white matter volumes with larger parietal/somatosensory white matter volumes than controls, particularly if UBO were present in the basal ganglia. All subjects with NF1 (including NF1+ADHD) had larger total and frontal white matter volumes than controls. Smaller frontal/right prefrontal gray matter volumes were found in NF1+ADHD when compared with NF1-pure patients. CONCLUSIONS: The increase in frontal and parietal white matter volumes in male patients with NF1, including the preferential centrencephalic distribution, supports the hypothesis that NF1's white matter pathology encompasses but is not limited to visible UBO. Male patients with NF1+ADHD, as compared with NF1-pure patients, showed frontal reductions that are largely consistent with those found in idiopathic ADHD.

Disproportionate increases of white matter in right frontal lobe in Tourette syndrome.

OBJECTIVE: Based on previous findings implicating abnormalities of cortico-striatal-thalamo-cortical circuitry in Tourette syndrome (TS), the authors performed a volumetric analysis of frontal and nonfrontal tissue (gray + white matter) in boys with TS, with and without attention deficit hyperactivity disorder (ADHD). METHODS: Frontal and nonfrontal gray and white matter compartment volumes, obtained by a MRI protocol, were analyzed with a 2 x 2 factorial multivariate analysis of variance approach for associations with a TS or ADHD factor in 11 boys with TS only, 14 with TS + ADHD, 12 with ADHD only, and 26 healthy boys. RESULTS: In subjects with TS, the right frontal lobe showed a larger proportion of white matter. In addition, results were consistent with previous reports of reduced frontal lobe volumes associated with ADHD. Our analyses suggested these reductions to be mainly the consequence of smaller gray matter volumes, particularly on the left. CONCLUSIONS: These findings, suggesting the volumetric composition of frontal lobe tissue to be different in TS, support the hypothesis proposing frontostriatal pathway involvement in the pathophysiology of the disorder. Differences in composition of right frontal lobe attributable to white matter do not definitively implicate the hypothesized fiber pathways; however, considered in the context of the unilateral directionality of frontal-striatal circuitry, these results suggest the white matter connections as one explanation for basal ganglia anomalies (loss of normal left > right asymmetry) in TS.

Neuroimaging studies in Rett syndrome.

Neuroimaging is a key instrument for determining structural and in vivo functional status of the brain, non-invasively. Multiple approaches can now determine aspects of anatomic and neurochemical changes in brain, and have been utilized effectively in Rett Syndrome patients to understand the biological basis of this neurodevelopmental disorder. Studies performed at our institute include volumetric analyses of MRI, magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI), cerebral blood flow measurements with MRI, and positron emission tomography scans (PET). These studies have provided considerable insight into mechanisms underlying the clinical features of this disease. Volumetric analyses suggest that decreased brain volume in RS results from global reductions in both gray and white matter of the brain. A selective vulnerability of the frontal lobes is evidenced by the preferential reduction of blood flow, increased choline and reduced n-acetyl aspartate (NAA) by MRS, and increased glucose uptake in these same regions as shown by ((18)F)-fluorodeoxyglucose (FDG) PET scans. We hypothesize that the increased glucose uptake relates to increased glutamate cycling in synapses. The resulting neuroexcitotoxic injury to the developing brain contributes to the seizures, behavioral disturbance and respiratory irregularities commonly seen in phases 1 and 2 of this disorder.

Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification.

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Age-dependent cognitive deficits and neuronal apoptosis in cyclooxygenase-2 transgenic mice.

The cyclooxygenases catalyze the rate-limiting step in the formation of prostaglandins from arachidonic acid and are the pharmacological targets of (NSAIDs). In brain, cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase, is selectively expressed in neurons of the cerebral cortex, hippocampus, and amygdala. As an immediate-early gene, COX-2 is dramatically and transiently induced in these neurons in response to NMDA receptor activation. In models of acute excitotoxic neuronal injury, elevated and sustained levels of COX-2 have been shown to promote neuronal apoptosis, indicating that upregulated COX-2 activity is injurious to neurons. COX-2 may also contribute to the development of Alzheimer's disease, for which early administration of NSAIDs is protective against development of the disease. To test the effect of constitutively elevated neuronal COX-2, transgenic mice were generated that overexpressed COX-2 in neurons and produced elevated levels of prostaglandins in brain. In cross-sectional behavioral studies, COX-2 transgenic mice developed an age-dependent deficit in spatial memory at 12 and 20 months but not at 7 months and a deficit in aversive behavior at 20 months of age. These behavioral changes were associated with a parallel age-dependent increase in neuronal apoptosis occurring at 14 and 22 months but not at 8 months of age and astrocytic activation at 24 months of age. These findings suggest that neuronal COX-2 may contribute to the pathophysiology of age-related diseases such as Alzheimer's disease by promoting memory dysfunction, neuronal apoptosis, and astrocytic activation in an age-dependent manner.

Annexin-1 is abnormally expressed in fragile X syndrome: two-dimensional electrophoresis study in lymphocytes.

The search for targets of FMRP (the product of FMR1, the mutated gene in Fragile X syndrome) has predominantly focused on identifying transcripts that are regulated by this RNA-binding protein. This study introduces the use of two-dimensional gel electrophoresis (2D PAGE) as a novel approach for demonstrating changes in protein synthesis secondary to FMRP deficit. By a standardized 2D PAGE protocol, we studied leukocyte homogenates from 30 males with different patterns of FMR1 mutation and different levels of FMRP. Samples from these subjects were compared to those of 12 normal control males and eight subjects with other mental retardation-associated conditions (i.e., Rett and Down syndromes). We found an abnormal pattern of a major leukocytic protein, identified by 2D PAGE datasets and immunoblotting as annexin-1 (Anx-1). Anx-1 appeared in subjects with Fragile X as multiple rather than 1-2 spots, at approximately 37 kd, in the pI 5-7 range. The presence and intensity of this Anx-1 pattern was relatively independent of Anx-1 levels and inversely related to total and high MW FMRP immunoreactivities. Based on the 2D PAGE pattern, without obvious MW change, and on dephosphorylation assays, we concluded that Anx-1's abnormality represents an aberrant posttranslational modification other than phosphorylation. Comparisons of our data with published cytoskeletal protein 2D profiles suggest that Anx-1 may be abnormally acetylated and, consequently, incapable of establishing appropriate N-terminal protein-protein interactions. In addition to its peripheral anti-inflammatory function, Anx-1 mediates glucocorticoid inhibition of the hypothalamo-pituitary-adrenal axis. As the latter seems to be disrupted in Fragile X syndrome, the reported Anx-1 abnormality could be responsible for some aspects of the Fragile X neurobehavioral phenotype. Our data also emphasize the feasibility of using 2D PAGE for disclosing molecular abnormalities in Fragile X and other genetic disorders.

Diffusion tensor imaging and axonal tracking in the human brainstem.

Diffusion tensor MRI was used to demonstrate in vivo anatomical mapping of brainstem axonal connections. It was possible to identify the corticospinal tract (CST), medial lemniscus, and the superior, medial, and inferior cerebellar peduncles. In addition, the cerebral peduncle could be subparcellated into component tracts, namely, the frontopontine tract, the CST, and the temporo-/parieto-/occipitopontine tract. Anatomical landmarks and tracking thresholds were established for each fiber and, using these standards, reproducibility of automated tracking as assessed by intra- and interrater reliability was found to be high (kappa > 0.82). Reconstructed fibers corresponded well to existing anatomical knowledge, validating the tracking. Information on the location of individual tracts was coregistered with quantitative MRI maps to automatically measure MRI parameters on a tract-by-tract basis. The results reveal that each tract has a unique spatial signature in terms of water relaxation and diffusion anisotropy.